Genomic Islands Identified in Highly Resistant Serratia sp. HRI: A Pathway to Discover New Disinfectant Resistance Elements

Molecular insights into the mechanisms of resistance to disinfectants are severely limited, together with the roles of various mobile genetic elements. Genomic islands are a well-characterised molecular resistance element in antibiotic resistance, but it is unknown whether genomic islands play a role in disinfectant resistance. Through whole-genome sequencing and the bioinformatic analysis of Serratia sp. HRI, an isolate with high disinfectant resistance capabilities, nine resistance islands were predicted and annotated within the genome. Resistance genes active against several antimicrobials were annotated in these islands, most of which are multidrug efflux pumps belonging to the MFS, ABC and DMT efflux families. Antibiotic resistance islands containing genes encoding for multidrug resistance proteins ErmB (macrolide and erythromycin resistance) and biclomycin were also found. A metal fitness island harbouring 13 resistance and response genes to copper, silver, lead, cadmium, zinc, and mercury was identified. In the search for disinfectant resistance islands, two genomic islands were identified to harbour smr genes, notorious for conferring disinfectant resistance. This suggests that genomic islands are capable of conferring disinfectant resistance, a phenomenon that has not yet been observed in the study of biocide resistance and tolerance.


Introduction
The COVID-19 pandemic has highlighted our need for effective disinfectants, antiseptics, and sanitisers (biocides). The antibiotic resistance crisis can be seen as a warning or foreshadowing of an equally alarming phenomenon of microbial resistance to disinfectants. This means it is troubling that, within the food and agricultural industries and medical environments, resistance to disinfectants amongst microorganisms is emerging at a startling rate [1][2][3][4].
Mobile genetic elements (MGEs) play a significant role in the transfer of genes which confer antimicrobial resistance [5][6][7][8]. Their mobility is brought about by horizontal gene transfer, resulting in populations with reduced susceptibility to various antimicrobials [5,8]. Resistance can develop against several antimicrobials simultaneously, without prior exposure [9]. Genomic island (GI) is an umbrella term for mobile genetic elements found on the bacterial chromosome that have been acquired through horizontal gene transfer, usually between 10 and 200 kb in length [6,10,11]. This overarching term also includes integrated plasmids, integrons, prophages, conjugative transposons, and integrative conjugative elements [6,[10][11][12]. These MGEs are then given more specific identities based on their mechanism of transfer (conjugation, transduction, or transformation) and genes present (transposases, integrases etc.) [6,12].
Genomic islands can be further characterised based on the phenotype they confer. For example, pathogenicity islands encode genes that confer an advantage in pathogenicity [13], resistance islands encode antimicrobial resistance genes [14], and metabolic islands contain genes that confer an additive metabolic advantage [6,10].
The bioinformatic identification of genomic islands is achieved using two approaches. The first is via sequence composition, and the second is via comparative genomics [10,11]. Both techniques have respective advantages and limitations, and therefore, a combination of the two provides the most sensitive and precise output [10,12]. IslandViewer4 is the gold standard for genomic island prediction, as it incorporates four different genomic island prediction methods, IslandPick, IslandPath-DIMOB, SIGI-HMM, and Islander [15].
Genomic islands have been found to play a role in antibiotic resistance [8,16]. However, minimal research has been carried out on the role of genomic islands in disinfectant resistance. As this is an emerging issue, more insight into the molecular mechanisms of resistance to disinfectants and other biocides is needed. A genomic island in Listeria monocytogenes isolates was found to be responsible for food-borne outbreaks harbouring multiple resistance genes, including an efflux pump involved in benzalkonium chloride resistance (ErmE) [17,18]. Jiang and co-workers (2020) found that the sug operon on the bacterial chromosome encoding SMR efflux pumps conferred resistance to benzalkonium chloride. This research brings forth the idea that resistance islands may be the latest genetic element capable of conferring resistance to disinfectants.
Resistance islands are often harboured in multidrug-resistant bacteria as one of many mechanisms to increase survivability [19]. One of these bacteria, Serratia sp. HRI, has high disinfectant resistance capabilities and provides a unique opportunity to study resistance to disinfectants and other biocides [20]. Several mechanisms of resistance to disinfectants have been elucidated, with efflux pumps being the most common. However, molecularbased resistance has mostly been limited to the study of plasmids. Little is known about which other mobile genetic elements can play a significant role in the development and dissemination of the disinfectant resistance phenotype. In the search for novel mechanisms of disinfectant resistance, genomic islands and the hypothetical proteins they harbour are attractive targets in the search for novel, previously undescribed mechanisms of resistance. If the molecular basis of disinfectant resistance is better understood, this will help to safeguard our current disinfectants and ensure proper biosafety in the agricultural, food, and medical industries. The aim of this work is to use prediction software and bioinformatic analysis to determine whether genomic islands can contribute to disinfectant resistance. The finding of several resistance islands harbouring known disinfectant resistance genes within this highly resistant isolate suggests that genomic islands can be characterised as a molecular element capable of conferring disinfectant and biocide tolerance and resistance. This paper adds to the evidence that genomic islands are capable of conferring biocide tolerance and resistance.

Materials and Methods
Serratia sp. HRI was isolated from a bottle of Didecyldimethylammonium chloride (DDAC)-based disinfectant [20]. Upon analysis, high levels of resistance to Quaternary Ammonium Compound (QAC) disinfectants were found via Minimal Inhibitory Concentration (MIC) tests [20].
The unusually high level of resistance observed in this isolate, together with its isolation from a bottle of disinfectant, prompted research into this microorganism. The genome of Serratia sp. HRI was sequenced and previously published [20]. The raw reads from this sequencing run, described previously, were then assembled again using the PATRIC (v. July 2021) de novo Genome Assembly service with default parameters unless otherwise specified (available at https://www.bv-brc.org/app/Assembly2) [21]. This assembled genome is 5 533 130 bp long, with GC content of 59.1%, an N50 score of 348 770, an L50 of 5, 47 contigs, and 126 RNAs, deposited on NCBI under Genbank Accession No. CP083690.1. This genome was uploaded to IslandViewer4 [15] with Serratia marcescens strain N4-5 chromosome sequence as a reference. IslandViewer4 uses four genomic island prediction methods (IslandPick, IslandPath-DIMOB, SIGI-HMM, and Islander) to identify genomic islands [15]. Thereafter, resistance genes are identified by IslandViewer4 using the Resistance Gene Identifier (RGI) from the Comprehensive Antibiotic Resistance Database (CARD) [22], as well as virulence factors from the Virulence Factor Database (VFDB) [23], PATRIC [24], and Victor's virulence factors (http://www.phidias.us/victors/ (accessed on 11 January 2022)), in addition to 18 919 pathogen-associated genes [25,26]. For further analysis and annotation, the sequence of each genomic island was uploaded to RAST and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) NCBI annotation tool for additional annotation [27,28].
In the GIs of interest (GI 11, 20, and 76), any gene annotated as a hypothetical or uncharacterised protein was finally run through the PSI-BLAST program [29] and annotated further if any significant hits were found.

Results
IslandViewer4 identified 92 genomic islands within the genome of Serratia sp. HRI, as depicted in Figure 1. Of the 92 genomic islands, 9 contained known antimicrobial resistance genes or genes implicated in antimicrobial resistance; these genomic islands were predicted via at least two prediction methods. Tables 1-4 represent the structure of these genomic islands and annotated gene lists [27,30,31]. This assembled genome is 5 533 130 bp long, with GC content of 59.1%, an N50 score of 348 770, an L50 of 5, 47 contigs, and 126 RNAs, deposited on NCBI under Genbank Accession No. CP083690.1. This genome was uploaded to IslandViewer4 [15] with Serratia marcescens strain N4-5 chromosome sequence as a reference. IslandViewer4 uses four genomic island prediction methods (IslandPick, IslandPath-DIMOB, SIGI-HMM, and Islander) to identify genomic islands [15]. Thereafter, resistance genes are identified by Is-landViewer4 using the Resistance Gene Identifier (RGI) from the Comprehensive Antibiotic Resistance Database (CARD) [22], as well as virulence factors from the Virulence Factor Database (VFDB) [23], PATRIC [24], and Victor's virulence factors (http://www.phidias.us/victors/ (accessed on 11 January 2022)), in addition to 18 919 pathogen-associated genes [25,26]. For further analysis and annotation, the sequence of each genomic island was uploaded to RAST and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) NCBI annotation tool for additional annotation [27,28].
In the GIs of interest (GI 11, 20, and 76), any gene annotated as a hypothetical or uncharacterised protein was finally run through the PSI-BLAST program [29] and annotated further if any significant hits were found.

Results
IslandViewer4 identified 92 genomic islands within the genome of Serratia sp. HRI, as depicted in Figure 1. Of the 92 genomic islands, 9 contained known antimicrobial resistance genes or genes implicated in antimicrobial resistance; these genomic islands were predicted via at least two prediction methods. Tables 1-4 represent the structure of these genomic islands and annotated gene lists [27,30,31].  Three of the nine genomic islands are shown in more detail as they contain resistance genes of particular interest (Tables 2-4); the remaining six islands are depicted in more detail in the Supplementary section (Tables S1-S6). Resistance island 11 is studied closely due to the number of resistance genes and their combination with hypothetical proteins, transcriptional regulators, and toxin-antitoxin systems. Resistance islands 20 and 76 are of interest as they contain known disinfectant resistance genes and a number of hypothetical proteins.
Genomic island 11 is represented in Table 2. This resistance island contains 78 annotated genes, including 7 genes encoding various efflux pumps. Of the seven genes, these include two copies of permeases of the drug/metabolite transporter (DMT) superfamily and a probable Co/Zn/Cd efflux system membrane fusion protein. Various components of efflux systems, such as an inner-membrane proton/drug antiporter (MSF type) of a tripartite multidrug efflux system, an outer membrane factor (OMF) lipoprotein, and two ABC-type antimicrobial peptide transport system proteins, make up the permease com-        Genes of interest in genomic island 20, represented in Table 3, include a small multidrug resistance efflux protein (SMR), an ABC transporter permease protein, and a probable Co/Zn/Cd efflux system membrane fusion protein. Several genes are associated with conjugative transfer, mobile element proteins, an integron-associated gene, and transposase-associated genes. Hypothetical protein 19 was further annotated by NCBI PGAP as an SMR family transporter, a well-known disinfectant resistance gene.
Genomic island 76, depicted in Table 4, contains 47 genes, including an smr gene and an ABC-type multidrug transport system gene, together with a complete toxin-antitoxin system (YoeB/YefM). This genomic island is also a mosaic of several mobile element associated genes, such as an integrase, repeat regions, recombinase, and multiple mobilisation proteins (MobA, MobC). Hypothetical protein 7 in GI 76 had a significant similarity hit in the BLAST program with a multidrug efflux ABC transporter permease/ATP-binding subunit SmdA (Max score: 25.0, Total score: 25.0, Query cover: 74%, E value: 1.9, Per. Ident: 26.51%). This protein is located next to a component of an ABC-type multidrug transport system and is likely part of an efflux system. Hypothetical protein 11, located adjacent to an SMR disinfectant resistance protein, had the highest similarity hit with GNAT family N-acetyltransferase (Serratia marcescens) when run through the BLAST program. This family of proteins is responsible for resistance to aminoglycoside antibiotics [32] and could play a role in the antimicrobial resistance of Serratia sp. HRI.
Although the following genomic islands were not highlighted, each has interesting characteristics and contains at least one antimicrobial resistance gene. Genomic island 18, depicted in Table S1 in the Supplementary section, contains heavy metal response genes to molybdenum and two ABC-type efflux pump permease components, YbhS and YbhR. These proteins, together with YbhF, form YbhFSR, which functions in tetracycline efflux and Na+(Li+)/H+ transport [33]. Adjacent to these genes is ybhL, a closely related gene whose function is unknown but is hypothesised to be involved in stress response and cell protection by unknown mechanisms [34]. Table S2 represents genomic island 23, which is one of the smallest GIs identified with only four genes. Some argue it should not be identified as a GI due to its small size [11]. However, as it contains a multidrug resistance gene from the DMT superfamily, it is noteworthy.
Genomic island 28, depicted in Table S3, contains genes encoding antibiotic multidrug resistance protein ErmB (macrolide and erythromycin resistance) and an adjacent ABC efflux gene [35,36]. This GI also contains multiple transposase genes and components from insertion sequence element IS911, suggesting this insertion sequence may have played a role in the evolution of this resistance island.
Genomic island 33 is a small island with only one annotated protein, shown in Table S4. The protein annotated is an HtpX protease, which, together with ClpA, is involved in aminoglycoside resistance in Stenotrophomonas maltophilia [37,38]. Although this island does not contain the ClpA gene, the HtpX protease has been co-selected with multiple hypothetical proteins, which may aid in its function and could be candidates for further study.
Genomic island 42 is a highly conserved metal response island, described in Table  S5, harbouring 13 genes involved in metal response with three complete toxin-antitoxin systems. Multiple toxin-antitoxin systems and several MGE-associated genes suggest this genomic island is mobile and highly conserved within a population. The toxin-antitoxin system, HigA/HigB, has been found to play a regulatory role in virulence and biofilm formation in Pseudomonas aeruginosa [39,40]. The metal response genes include those for silver and copper, which are being promoted as used in some products an alternatives to current antimicrobials [41]. These characteristics threaten the efficacy of the potential of this alternative treatment.
A bicyclomycin resistance protein can be found on genomic island 46 in Table S6. This resistance protein, together with error-prone repair (UmuD) and error-prone DNA polymerase (UmuC), could introduce mutations and aid in the evolution of antimicrobial resistance.

Discussion
Resistance islands are a well-known molecular element capable of conferring antibiotic resistance [42], but little research has been carried out on whether these mobile elements play a role in disinfectant and biocide resistance. Improved sequencing technology and more accessible bioinformatic programs have opened the door to the study of these ele-ments and their impact on the resistance profile. This work aims to use these advances in sequencing technology to identify regions likely characterised as resistance islands contributing to the high levels of disinfectant resistance observed in this isolate.
These results are integrated images and gene annotations generated by the Island-Viewer4, RAST, PGAP, and PSI-BLAST programs. A total of 92 genomic islands were found within the genome of Serratia sp. HRI, and a few are highlighted here as they are of extrachromosomal origin, identified within a highly resistant microorganism, and harbour antimicrobial resistance genes. The vast amount of genomic islands identified within Serratia sp. HRI aligns with the predicted high level of plasticity within the Serratia genus [5]. High genomic plasticity can lead to a mosaic of MGEs and can be attributable to resultant antimicrobial resistance [8]. Iguchi and co-workers (2014) found high genome plasticity in a clinical Serratia marcescens isolate. Compared to a non-resistant isolate, a mosaic of mobile genetic elements and acquired resistance genes contributed to the high levels of antimicrobial resistance in the clinical isolate [5].
Genomic island 11 was the first presented here and can be described as an all-round resistance and fitness island, as it harbours several annotated resistance genes applicable to various antimicrobials. This genomic island includes partial efflux systems from the MFS, OMF, and ABC families and two copies of complete systems from the DMT efflux family. Efflux genes that are not labelled as resistance genes are also highlighted, as they are part of the genome of a highly resistant isolate, placed within a resistance island, and close to a resistance efflux system. Therefore, they are of interest for further study. This genomic island also carries genes involved in metal response, colicin immunity, transcriptional regulators, and multiple MGE components (insertion sequences, phage integrase, and mobility genes). All four transcriptional regulator families found within this GI have been shown to improve bacterial fitness and survivability. LysR-type transcriptional regulators have been reported to play a role in antibiotic resistance in Aeromonas sp. [43]. LuxR transcriptional regulators are involved in biofilm formation and stress response in Pseudomonas and Mycobacterium sp. [44,45]. AcrR transcriptional regulators and their mutations have been seen to contribute towards drug resistance in Salmonella sp. [46]. Finally, the possible regulatory protein thioredoxin (Trx) protects against oxidative stress, a well-established response after treatment by antimicrobials such as disinfectants [47]. Interestingly, more than half of all the genes present in this island are uncharacterised and are listed as hypothetical proteins. As this is a large genomic island and requires metabolic resources to maintain and transcribe these elements, it is intriguing that these genes have not been lost. This suggests that some of these hypothetical proteins which form the majority of this genomic island may have a function and are attractive candidates in the search for novel resistance genes and even novel mechanisms of resistance.
Genomic island 20 contains the first gene directly implicated in disinfectant resistance, the smr gene [19,48], as well as an ABC efflux permease protein. This island also contains a metal response gene and multiple conjugative transfer proteins alluding to the origin of this GI. Within this sequence, a mosaic of MGEs, including genes encoding transposases, an integrase, and mobile element proteins, were discovered. Multiple transcription regulators associated with antimicrobial resistance are again present in this GI, including regulators from the LysR family and Tetr families, linked to tetracycline resistance [49,50]. Within this resistance island, 11 out of 41 genes are uncharacterised and annotated as hypothetical proteins. This island contains multiple MGEs, suggesting high plasticity, and the probability of incorporating additional resistance determinants is high.
Genomic island 76 contains a complete toxin-antitoxin system (Yoe-B/YefM), an ABC multidrug efflux-encoding gene and, importantly an smr gene. This resistance island is conservable in a population due to the toxin-antitoxin system, and almost two-thirds of the genes in this island are uncharacterised. Out of the 47 genes making up this GI, 29 are hypothetical proteins that have been co-selected and maintained with the antimicrobial resistance genes in this island. These uncharacterised flanking sequences are potential targets in the search for new mechanisms of resistance.
When considered all together, these genomic islands contain multiple antimicrobial resistance genes harboured simultaneously within the genome of Serratia sp. HRI, which can confer a wide range of resistance within this single isolate. Although there were many incomplete efflux systems ( GIs 11,18,19,20,28, and 76), bioinformatics and annotation software still have a way to go, and in the years to come, these systems may be annotated differently.
In a field such as disinfectant resistance, where knowledge of mechanisms is minimal, the vast numbers of hypothetical proteins within these resistance islands are attractive targets in searching for novel resistance genes and mechanisms of disinfectant resistance.
It is also interesting that very few genes identified in these islands were assigned to subsystems after annotation. This adds to the notion that bioinformatics and annotation programs need improvement, as more information is needed on where these genes fit into the bacterial metabolism and their function(s).
The plasticity and adaptability of the Serratia genome shows the capability of the this genus in acquiring MGEs that can contribute to the decreased susceptibility often observed in the Serratia genus [5]. The result is observed in isolates such as Serratia sp. HRI, whose genome is an assortment of fitness determinants gathered over time, increasing survivability to a wide range of antimicrobials. To confirm the phenotypic impact of these resistance islands and the extent of their impact, further work will be required.

Conclusions
There is limited information on whether genomic islands are capable of conferring resistance to disinfectants. Therefore, the genomic islands of Serratia sp. HRI will add to the knowledge of antimicrobial resistance and reinforce the idea that genomics islands can be described as the latest molecular element capable of conferring disinfectant resistance. This work also adds to the evidence for the cross-resistance and co-selection of antimicrobial resistance genes within a single organism. This work represents how predictive bioinformatic technology can lead targeted research into antimicrobial resistance. However, this is a starting point and only tells scientists where to look instead of providing a definitive answer. Phenotypic analysis needs to be coupled with predictive software to fully elucidate resistance mechanisms.
The increased use of disinfectants during the COVID-19 pandemic will inevitably give rise to less susceptible populations at an advanced rate. Amidst the pandemic, we are silently and unknowingly selecting disinfectant-resistant microorganisms. By getting ahead of disinfectant resistance, we will be able to safeguard our current disinfectants and ensure infection control in both the agricultural and medical industries.